Mitochondrial aldehyde dehydrogenase ALD5 of Saccharomyces cerevisiae is involved in the biosynthesis of mitochondrial electron transport chain, and the ald5 mutant is incompetent for respiration. With use of the mutant, we examined the detoxication of H2O2 generation by fatty acid beta-oxidation in peroxisome. The ald5 mutant (AKD321), as well as the 746 rho0 mutant, was more resistant to H2O2 stress than the wild type. However, overexpression of the MDH3 gene that was involved in the reoxidation of NADH during fatty acid beta-oxidation caused a decrease in cell viability of AKD321 to H2O2 stress, while the 746 rho0 mutant had no such effect. Intracellular H2O2 concentration increased approximately fourfold in MDH3 overexpressing ald5 strain (MD3-AKD321), compared with AKD321. The peroxisomal catalase activity of MD3-AKD321 decreased by 83% to that of AKD321. And also, the overexpression of MDH3 had only a weak effect in MDH3 overexpressing 746 rho0 strain, decreasing by 14% to that of 746 rho0 mutant. The increased palmitoyl CoA oxidation by overexpression of MDH3 gene was the same in both strains. Under conditions of MDH3 overexpression, peroxisomal catalase (CTA1) appears to be a limiting factor to oxidative stress. These observations point to an important, as yet unidentified, role of mitochondrial aldehyde dehydrogenase (ALD5) to endogeneous oxidative stress in peroxisome.